The biotransformation of steroids by conjugation to a highly charged sulfonate group is a functionally important process that results in a marked change in the physicochemical properties of these essentially hydrophobic compounds. This fact notwithstanding, the biological significance of steroid sulfonation is poorly understood. The transfer of a sulfonate group (SO3-) to an appropriate acceptor site is carried out by enzymes termed sulfotransferases. By altering steroid polarity, sulfonation increases water solubility and modifies protein binding, thereby influencing steroid transportability and acting as an intracellular trapping or storage mechanism. Furthermore, by serving to either initiate an event (steroid sulfonate is the active form) or terminate an event (steroid sulfonate is the inactive form), sulfonation can function to regulate hormonal responsivity. One of the most active tissues to sulfonate steroids is the adrenal cortex; furthermore, specific steroid sulfotransferases are differentially expressed in functionally distinct adrenocortical zones. To seek insight into the molecular mechanisms and biological consequences of steroid sulfonation, we are currently investigating steroid-specific sulfotransferases as well as enzymes responsible for the production of the universal sulfonate donor (PAPS). Projects include: 1) isolation, characterization, antibody production, and cDNA cloning and expression of specific enzymes; 2) genomic cloning and assessment of transcriptional regulation; 3) mutational studies to identify amino acid residues that create steroid substrate and sulfonate donor sites of interaction; 4) experiments regarding the structural basis for chiral specificity. The complete estrogen sulfotransferase (EST) gene, including the 5'-flanking region, has now been cloned and examination of its transcriptional regulation by steroid hormones is ongoing. An amino acid motif near the C-terminus of EST that is critical to binding of the sulfonate donor molecule has been identified and amino acid residues involved in estrogen interaction with EST have been specified by site-directed mutagenesis. The cDNA for chiral-specific 3b-hydroxysteroid sulfotransferase (HST) has been cloned and expressed, and structural genes for 3a- and 3b-HST have been partially cloned.